PROJECT SUMMARY Currently, no effective therapy exists to fully recover lost tissue or function following an acute severe ischemic event. Cell replacement therapy provides a promising solution for patients that suffer from loss of tissue responsible for functions such as motor movement following a stroke. Previous transplant studies of human pluripotent stem cell-derived neurons (hPSNs) in a rodent model of stroke have not revealed significant anatomical integration or true cell replacement long-term. Understanding whether true cell replacement is possible and necessary for long-term recovery of function is important for the field to progress. The overall objective of this fellowship proposal is to determine whether directed differentiation of a specific hPSN phenotype will improve anatomical integration with host tissue following stroke. The central hypothesis to be tested is that promoting targeted differentiation of corticofugal projection neurons will improve long-term integration with appropriate host tissue. The following specific aims will be used to test the hypothesis: Specific Aim 1: Directed differentiation of hPSNs to corticofugal projection neurons (CFuPNs) in vitro. Hypothesis: hPSNs can be directed to a layer V corticofugal phenotype by utilizing virus-induced overexpression of an identified master regulator, FEZF2. Specific Aim 2: Determine if a specified population of hPSNs will structurally integrate more effectively in healthy and injured tissue. Hypothesis: Directed corticofugal projection hPSNs can form appropriate anatomical connection in vivo in healthy and injured host environments. Experiments are designed to 1) determine whether a single master regulator can drive differentiation of a scalable human stem cell population to a critical neuronal population that is lost during stroke, and 2) to determine whether this cellular phenotype is superior to default differentiation at promoting specific anatomical reconstruction following transplantation in healthy and injured environments. We will specifically identify whether FEZF2 can drive differentiating neurons to become layer V corticofugal projection neurons, and whether these send neuronal fibers to subcortical white matter tracts such as internal capsule and pyramids after transplantation in healthy and injured tissue. This will reveal whether deriving a specific phenotype of cells lost during an injury, such as stroke, is relevant to promoting integration of these cells with host tissue. Successful completion of proposed aims will significantly further the field by revealing an improved model to study true cell replacement for stroke and push other fate-specific phenotypes to be investigated for other neuropathological events leading to tissue loss.